PEGylated peptides and other macromolecules —i.e. macromolecules bearing a polyethylene glycol (PEG) substituent—are widely used, in particular for the preparation of pharmaceuticals, drug delivery vehicles, and other biocompatible materials.
For the purposes of pharmaceutical development, the most common conjugation reaction is the addition of thiols (i.e. cysteine side chains) to a drug, or PEG reagent bearing an electrophilic moiety such as a maleimide or alpha-halo amide. Other solutions include oxime formation (the reaction of a hydroxylamine with a ketone or aldehyde) and non-specific amide formation on lysine side chains. Chemoselective conjugations using the triazole forming reactions of azides and terminal alkynes are also popular, at least in academic research. The main limitations are either the need for toxic copper reagents or slow rate constants requiring an excess of one of the reagents.
Since both the macromolecules and the polyethylene glycol substituents tend to be expensive and complex in preparation, it is important that the PEGylation is selective and highly efficient. Furthermore, it is desirable that a stable natural bond is formed, i.e. a bond that is commonly encountered in naturally occurring materials and which is known to be stable and safe for use in medical and materials applications.
Chemical reactions that allow selective bond formation between two reactants even in the presence of many unprotected functional groups are important but rare. The ideal chemoselective conjugation reaction would allow rapid covalent bond formation between two unique but chemically stable moieties under aqueous conditions using equimolar amounts of the ligation partners, regardless of the size of the substrate or number and nature of unprotected functional groups.
Such feats of bond construction, such as DNA ligation, are routinely accomplished by specific biochemical enzymes and can, in certain circumstances, be co-opted for synthetic applications. But fast, selective strictly chemical ligations are so far unknown. The few known synthetic ligations form unnatural bonds, require the presence of toxic reagents, or do not proceed fast enough to conjugate equimolar quantities of large or valuable starting materials.
Therefore, one of the remaining challenges in this area is the identification of faster ligations (second order rate constants >10 M−1 s−1) that form natural bonds, preferably under aqueous conditions, without added reagents or catalysts.
More general, the technical problem to be solved by the present invention is the selective formation of a covalent bond between two large molecules. This technical problem occurs frequently in the synthesis of biologically active molecules including, but not limited to, proteins, peptides, PEGylated biomolecules and peptides, antibody drug conjugates, and functionalized polymers. Typical reactions for covalent bond formation suffer from three problems that make this difficult:                a. Most organic reactions require anhydrous, organic solvents and do not operate properly in the presence of unprotected organic functional groups (i.e. amines, carboxylic acids, alcohols, thiols, etc.).        b. Most organic reactions for joining large molecules are too slow, requiring high concentrations (10 mM or higher), long reaction times, a large excess of one reactant, and/or high temperatures.        c. The best known solutions for joining two large, unprotected molecules give an unnatural connectivity that can be problematic, immunogenic, or which cannot be used to make the natural forms of molecules such as proteins.        
In order to join large molecules using equimolar amounts of each reactant—which is important for economic and purification reasons—a second order rate constant of at least 1 M−1 s−1 is required. Faster rate constants are even more desirable.